The Journal of Membrane Biology

, Volume 192, Issue 3, pp 169–179 | Cite as

Coupling of CFTR-mediated anion secretion to nucleoside transporters and adenosine homeostasis in calu-3 cells

  • A. J. Szkotak
  • A. M. L. Ng
  • S. F. P. Man
  • S. A. Baldwin
  • C. E. Cass
  • J. D. Young
  • M. Duszyk
Article

Abstract

The purpose of this study was to characterize the role of adenosine-dependent regulation of anion secretion in Calu-3 cells. RT-PCR studies showed that Calu-3 cells expressed mRNA for A2a and A2B but not A1 or A3 receptors, and for hENTl, hENT2 and hCNT3 but not hCNTl or hCNT2 nucleoside transporters. Short-circuit current measurements indicated that A2b receptors were present in both apical and basolateral membranes, whereas A2a receptors were detected only in basolateral membranes. Uptake studies demonstrated that the majority of adenosine transport was mediated by hENTl, which was localized to both apical and basolateral membranes, with a smaller hENT2-mediated component in basolateral membranes. Whole-cell current measurements showed that application of extracellular nitrobenzylmercaptopurine ribonucleoside (NBMPR), a selective inhibitor of hENTl-mediated transport, had similar effects on whole-cell currents as the application of exogenous adenosine. Inhibitors of adenosine kinase and 5′-nucleotidase increased and decreased, respectively, whole-cell currents, whereas inhibition of adenosine deaminase had no effect. Single-channel studies showed that NBMPR and adenosine kinase inhibitors activated CFTR Cl- channels. These results suggested that the equilibrative nucleoside transporters (hENTl, hENT2) together with adenosine kinase and 5′-nucleotidase play a crucial role in the regulation of CFTR through an adenosine-dependent pathway in human airway epithelia.

Key words

Calu-3 cells Adenosine metabolism Nucleoside transport CFTR 

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References

  1. Avila, M.Y., Stone, R.A., Civan, M.M. 2001. A1-, A2A- and A3- subtype adenosine receptors modulate intraocular pressure in the mouse. Br. J. Pharmacol. 134:241–245PubMedCrossRefGoogle Scholar
  2. Baldwin, S.A., Mackey, J.R., Cass, C.E., Young, J.D. 1999. Nucleoside transporters: molecular biology and implications for therapeutic development. Mol. Med. Today 5:216–224PubMedCrossRefGoogle Scholar
  3. Blackburn, M.R., Volmer, J.B., Thrasher, J.L., Zhong, H., Crosby, J.R., Lee, J.J., Kellems, R.E. 2000. Metabolic consequences of adenosine deaminase deficiency in mice are associated with defects in alveogenesis, pulmonary inflammation, and airway obstruction. J. Exp. Med. 192:159–170PubMedCrossRefGoogle Scholar
  4. Cobb, B.R., Ruiz, F., King, CM., Fortenberry, J., Greer, H., Kovacs, T., Sorscher, E.J., Clancy, J.P. 2002. A2 adenosine receptors regulate CFTR through PKA and PLA2 Am. J. Physiol. 282:L12-L25Google Scholar
  5. Crawford, C.R., Patel, D.H., Naeve, C, Belt, J.A. 1998. Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line. J. Biol. Chem. 273:5288–5293PubMedCrossRefGoogle Scholar
  6. Deussen, A. 2000. Metabolic flux rates of adenosine in the heart. Naunyn Schmiedebergs Arch. Pharmacol. 362:351–363PubMedCrossRefGoogle Scholar
  7. Felipe, A., Valdes, R., Santo, B., Lloberas, J., Casado, J., Pastor-Anglada, M. 1998. Na+-dependent nucleoside transport in liver; two different isoforms from the same gene family are expressed in liver cells. Biochem. J. 330:997–1001PubMedGoogle Scholar
  8. Fredholm, B.B., Arslan, G., Halldner, L., Kull, B., Schulte, G., Wasserman, W. 2000. Structure and function of adenosine receptors and their genes. Naunyn Schmiedebergs Arch. Pharmacol. 362:364–374PubMedCrossRefGoogle Scholar
  9. Griffiths, M., Beaumont, N., Yao, S.Y., Sundaram, M., Boumah, C.E., Davies, A., Kwong, F.Y., Coe, I, Cass, C.E., Young, J.D., Baldwin, S.A. 1997a. Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs. Nat. Med. 3:89–93PubMedCrossRefGoogle Scholar
  10. Griffiths, M., Yao, S.Y., Abidi, F., Phillips, S.E., Cass, C.E., Young, J.D., Baldwin, S.A. 1997b. Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. Biochem. J. 328:739–743PubMedGoogle Scholar
  11. Hamilton, S.R., Yao, S.Y.M., Ingram, J.C., Hadden, D.A., Ritzel, M.W.L., Gallagher, M.P., Henderson, P.J.F., Cass, C.E., Young, J.D., Baldwin, S.A. 2001. Subcellular distribution and membrane topology of the mammalian concentrative Na+ -nucleoside cotransporter rCNTl. J. Biol. Chem. 276:27981–27988PubMedCrossRefGoogle Scholar
  12. Huang, P., Lazarowski, E.R., Tarran, R., Milgram, S.L., Boucher, R.C., Stutts, M.J. 2001. Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells. Proc. Natl. Acad. Sci. USA 98:14120–14125PubMedCrossRefGoogle Scholar
  13. Leung, G.P.H., Ward, J.L., Wong, P.Y.D., Tse, CM. 2001. Characterization of nucleoside transport systems in cultured rat epididymal epithelium. Am. J. Physiol. 280:C1076-C1082Google Scholar
  14. Mun, E.C., Tally, K.J., Matthews, J.B. 1998. Characterization and regulation of adenosine transport in T84 intestinal epithelial cells. Am. J. Physiol. 274:G261-G269PubMedGoogle Scholar
  15. Musante, L., Zegarra-Moran, O., Montaldo, P.G., Ponzoni, M., Galietta, LJ. 1999. Autocrine regulation of volume-sensitive anion channels in airway epithelial cells by adenosine. J. Biol. Chem. 274:11701–11707PubMedCrossRefGoogle Scholar
  16. Poulsen, S.A., Quinn, R.J. 1998. Adenosine receptors; new opportunities for future drugs. Bioorg. Med. Chem. 6:619–641PubMedCrossRefGoogle Scholar
  17. Ralevic, V., Burnstock, G. 1998. Receptors for purines and pyrimidines. Pharmacol. Rev. 50:413–492PubMedGoogle Scholar
  18. Ritzel, M.W., Ng, A.M., Yao, S.Y., Graham, K., Loewen, S.K., Smith, K.M., Hyde, R.J., Karpinski, E., Cass, C.E., Baldwin, S.A., Young, J.D. 2001. Recent molecular advances in studies of the concentrative Na+-dependent nucleoside transporter (CNT) family: identification and characterization of novel human and mouse proteins (hCNT3 and mCNT3) broadly selective for purine and pyrimidine nucleosides (system cib). Mol. Membr. Biol. 18:65–72PubMedCrossRefGoogle Scholar
  19. Spychala, J., Datta, N.S., Takabayashi, K., Datta, M., Fox, I.H., Gribbin, T., Mitchell, B.S. 1996. Cloning of human adenosine kinase cDNA: Sequence similarity to microbial ribokinases and fructokinases. Proc. Natl. Acad. Sci. USA 93:1232–1237PubMedCrossRefGoogle Scholar
  20. Szkotak, A.J., Ng, A.M.L., Sawicka, J., Baldwin, S.A., Man, S.F.P., Cass, C.E., Young, J.D., Duszyk, M. 2001. Regulation of K current in human airway epithelial cells by exogenous and autocrine adenosine. Am. J. Physiol. 281:C1991-C2002Google Scholar
  21. Taira, M., Tamaoki, J., Nishimura, K., Nakata, J., Kondo, M., Takemura, H., Nagai, A. 2002. Adenosine A3 receptor-mediated potentiation of mucociliary transport and epithelial ciliary motility. Am. J. Physiol. 282:L556-L562Google Scholar
  22. Wagner, D.R., Bontemps, F., van den, B.G. 1994. Existence and role of substrate cycling between AMP and adenosine in isolated rabbit cardiomyocytes under control conditions and in ATP depletion. Circ. Res. 90:1343–1349Google Scholar
  23. Ward, J.L., Tse, CM. 1999. Nucleoside transport in human colonic epithelial cell lines: evidence for two Na+-independent transport systems in T84 and Caco-2 cells. Biochim. Biophys. Acta 1419:15–22PubMedCrossRefGoogle Scholar
  24. Wu, X., Yuan, G., Brett, CM., Hui, A.C., Giacomini, K.M. 1992. Sodium-dependent nucleoside transport in choroid plexus from rabbit. Evidence for a single transporter for purine and pyrimidine nucleosides. J. Biol. Chem. 267:8813–8818PubMedGoogle Scholar
  25. Yao, S.Y.M., Ng, A.M.L., Vickers, M.F., Sundaram, M., Cass, CE., Baldwin, S.A., Young, J.D. 2002. Functional and molecular characterization of nucleobase transport by recombinant human and rat equilibrative nucleoside transporters 1 and 2. Chimeric constructs reveal a role for the ENT2 helix 5-6 region in nucleobase translocation. J. Biol. Chem. 277:24938–24948PubMedCrossRefGoogle Scholar
  26. Young, J.D., Cheeseman, C.I., Mackey, J.R., Cass, C.E., Baldwin, S.A. 2000. Molecular mechanisms of nucleoside and nucleoside drug transport. In : Current Topics in Membranes. K.E. Barrett and M. Donowitz, editors, pp. 329–379 Academic Press, San Diego, CAGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • A. J. Szkotak
    • 1
  • A. M. L. Ng
    • 1
  • S. F. P. Man
    • 1
  • S. A. Baldwin
    • 3
  • C. E. Cass
    • 2
  • J. D. Young
    • 1
  • M. Duszyk
    • 1
  1. 1.Membrane Protein Research Group, Departments of PhysiologyUniversity of AlbertaEdmontonCanada
  2. 2.Membrane Protein Research Group, Departments of OncologyUniversity of AlbertaEdmontonCanada
  3. 3.School of Biochemistry and Molecular BiologyUniversity of LeedsLeedsUK

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